Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure

• Published in: Biochemistry (Easton) 1987-01, Vol.26 (1), p.5-9
• Main Authors: VANDAELE, S, FOSSET, M, GALIZZI, J.-P, LAZDUNSKI, M
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal; Biological and medical sciences; Calcium - metabolism; Calcium Channel Blockers - metabolism; Calcium Channels; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; Ion Channels - metabolism; Kinetics; Microtubules - metabolism; Molecular and cellular biology; Molecular Weight; Muscles - metabolism; Peptide Mapping; Rabbits; Receptors, Nicotinic - immunology; Receptors, Nicotinic - isolation & purification; Receptors, Nicotinic - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00375a002

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.